Process for preparing oestrogenic hormones from urine



May 1, 1951 A. s. CQOK ETAL 2,551,205

PROCESS FOR PREPARING OESTROGENIC HORMONES FROM URINE Filed oct. 1, 1947v SUL VEA/ @il EURHTO/v WH TE-/A/S UBLE 50.1. VENT fum/E PR//vf/PLES(wam/P0) iatented May l,

PROCESS FOR. PREPARING OESTRGENIC HORMONES FROM URINE Arthur StanleyCook and Gordon A. Grant, Montreal, Quebec, Canada, assignors to Ayerst,Mc- Kenna & Harrison, Limited, Montreal, Quebec, Canada, a corporationof the Dominion of `Canada Application `October 1, 1947, Serial No.777,370

(Cl. IS7-74.5)

w Claims.

1 Introduction This invention relates to Water-soluble oestrogenichormone preparations derived from equine urine, for example, that of astallion or pregnant mare. This application is a continuation-in-part ofapplication S. N. 536,960 filed May 23, 1944, now Patent No. 2,429,398.The product of the invention is distinguished generally from the manyWater-insoluble oestrogenic hormone preparations by its substantialinsolubility in water-insoluble organic solvents such as benzol andether, its substantial solubility in Water, its high stability instorage, therapeutic nontoxicity, and the ability to produce vaginalcornication in adult ovariectomized rats and to alleviate the menopausalsyndrome in humans.

Clinically applicable Water-soluble substances having the abovecharacteristics and high Oral oestrogenic activity may, for example, bederived by concentrating urine, while still fresh and thus unhydrolyzed,or suitably preserved to prevent hydrolysis. This concentration mayconveniently be accomplished by adsorption with an adsorbent andsubsequent elution with a watersoluble organic solvent. The adsorbentmaterial may be charcoal, fullers earth, calcium triphosphate or anadsorbent clay, with charcoal preferred. The eluting agent may be anorganic nitrogen-containing base by choice, pyridine or a mixture of analcohol and chloroform. Then, the eluate is concentrated by removal ofthe solvent and the largely aqueous concentrate so obtained ispreferably treated With a water-immiscible organic solvent such asbenzol and/or ether, so as to remove substances soluble in the saidsolvent. This procedure is taught in our Patent No. 2,429,- 398.

The important aspects of the patented procedure described are that thestarting material is unhydrolyzed and that conditions are maintainedthroughout the process to prevent hydrolysis of the active principles.For example, the temperature is preferably maintained at a relativelylow level of say from about 40C. to about 50 C. The time of treatment atabove room temperature is kept as short as possible and the pH ismaintained Within the neutral to mildly alkaline range, say from about'7 to about 8.5.

The resulting product of the said patent Was an aqueous concentrate(containing about solids) or, if dried, a Water-soluble powdercontaining substantially all the Water-soluble oestrogenic activity andother therapeutic principles of the original urine and generally fromabout l to about 2% of the original urinary solids.

The product contained substances rendering it stable and at the sametime, it was free from toxicity in a therapeutic sense when used atdosage levels satisfactory to alleviate the symptoms of the menopausalsyndrome.

However, when necessary to administer orally Such oestrogenic materialat a very high dose level in particular therapy, it has been found thatthe above :preparation may in certain cases cause nausea and othergastric disturbances. The applicants have now discovered that thefactors responsible for such disturbances can be removed withoutsubstantial loss of oestrogenic and other therapeutic principles andwithout loss of stability.

It has been found that compounds which cause nausea and other gastricdisturbances are certain fats, oils, phenols, alkyl-substituted phenols,etc., which are present as salts. To remove these substances, theconcentrated oestrogenic material is brought to an acid pH thusreleasing or liberating the oils, fats and free bases and this isfollowed by solvent Washing using a solvent in which the toxic oils andphenolic substances are selectively soluble while the desiredoestrogenic compounds are substantially insoluble.

According to a preferred embodiment of the present invention the aqueousconcentrate obtained by concentration of the Water-solvent eluate asdescribed above and in Patent No. 2,429,398 is treated as follows. ThepH is adjusted to within the range from about 2 to about 6, preferablyfrom about 4 to about 5. The concentrate is then washed with awater-immiscible organic solvent, the temperature being maintainedpreferably Within the range from about 15 C. to about 20 C., althoughother conditions being propitious, this temperature can rise to 40 C.The washing or extraction is carried out as fast as possible, theactivity remaining in the aqueous phase. The important thing is tocoordinate the conditions so as to prevent hydrolysis.

An aqueous extract containing the water-soluble oestrogenic substancesand other therapeutic principles and free from much of the bulk andundesirable impurities is thus obtained. The extract is then neutralizedto a pH from about '7 to about 8. At this stage it can be Washed with anether to remove excess solvent, but this is an optional step. Theneutralized extract is then dried, preferably by distillation in vacuoat a temperature within the range from about 40 C. to about 50 C., toform a substantially dry residue.

This residue has surprisingly high oral oestrogenic activity and ischaracterized by the absence of substantially water-immisciblesolvent-soluble substances which are odoriferous and produce gastricdisturbances and nausea in certain individuals. This product is capableof clinical use, but the applicants prefer to process it further as willbe described. Further, crude products may be derived by processing inaccordance With the teachings-of `the present application, crudermaterials than the preferred starting materials above dened. e

The oestrogenically active residue is dissolved in a small amount of alower `alcohol and the solution treated with a ketonic solvent to form aprecipitate containing very littleof the activity. Further quantities ofthe odoriferous and toxic factors characteristic of crude' urineconcentrates, and inert solids are thus removed inthe precipitate, whichcontains little oestrogenic activty. The supernatant liquid containingthe activity in solution in the alcohol-ketone mixture is separated andconcentrated to dryness.l "The dried residue may againv be dissolved ina lower alcohol and the active principles'precipitated by the additionof an ether. This removed in the extracting solution more undesirablefactors particularly toxic substances "and oils. The precipitate isseparated and washed with an ether and then dried. The dried precipitatecontains a large proportion of the water-soluble oestrogenic substancesof the starting concentrate. It might be explained that where a loweralcohol is mentioned, one containing 5 carbon atoms or less is intended,with methyl or ethyl alcohol being preferred.

The water-immiscible organic solvent extraction and ketonicprecipitation steps have the unexpected efect of removing inert solids,toxic and odoriferous substances, While not reducing to nearly theextent which might reasonably ybe expected the water-soluble oestrogenicactivity or removing or inactivating factors producing stability. `Theapplicants product differs from pure oestrone sulphate by its stabilityand enhanced therapeutic effect. Thus a feature of the process is theability to yield a product containing watersoluble oestrogenicsubstances in a concentrated yet stable form.

While the applicants have described a preferred procedure in which thewater-insoluble solvent washing is used first, and the ketonicprecipitation later, it is also possible to reverse the order of thesesteps. Other modications are also `possible within the general teachingsof the present application.

The invention will be more completely understood by reference to thefollowing examples illustrating preferred proceduresv and to thedrawing.

The drawing shows a flow sheet which represents a complete processtogether with a modication thereof.

Example 1 About 25 gallons of urine freshly obtained from pregnant maresand containing water-soluble oestrogenic activity were preserved with analcohol-chloroform mixture.

About 560 grams of activated carbon was added and the mixture stirredmechanically for about 15 minutes at a suitable temperature, e. g.between about 15Q C. and about 30 C. The carbon was filtered off. Theurine filtrate was re-treated with carbon. The carbon adsorbing agentscontaining the oestrogenic activity were combined. Substantially all thewater-soluble oestrogenic substances had been removed from the urine bythis adsorption step. In one procedure the carbon was then dried, inanother it was left wet,

e. g. the charcoal can be used right after filtration or it can be driedand used later.

About three liters of an aqueous solution containing about pyridine wasadded to the carbon (dry or wet) and mixed thoroughly at about roomtemperature. This mixture was stirred and then allowed to stand in therefrigerator several hours. The carbon was then filtered off andresuspended in about two liters of fresh pyridine solution (about 90%).It was again allowed to stand for several hours with occasionalstirring. The carbon was then filtered oli. The carbon was suspendedagain in about two liters of pyridine solution and then sucked dry.

The pyridine extract and washings obtained in the above manner,amounting now to about eight liters were combined and concentrated invacuo at a low temperature between about 40 C. and about 50C. to 250cubic centimeters. During the final stage of preparation in which thepyridine was being removed and the largely aqueous concentrate obtained,the reaction of the mixture was not allowed to become acid. About 250cubic centimeters of water were added. This solution was a concentrateand contained a substantial proportion of the water-soluble oestrogenicsubstances of the original urine.

About 400 ml. of the concentrate, prepared as described above,containing water-soluble oestrogenie potency equivalent to 19 mg. sodiumoestrone sulphate per ml. (as determined by the modified Marrian-Kobertest) was acidied to pH 4 with 15% aqueous sulphuric acid and extractedfour times with 160 ml. of ethylene dichloride (each extraction) andthree times with 80 ml. of ethylene dichloride (each extraction). Thesolvent extracts were discarded. The aqueous concentrate was thenadjusted to pI-l 6.9 with 5 N sodium hydroxide and extracted three timeswith ml. (each extraction) of ether. The aqueous concentrate thus freedfrom odoriferous oils, phenols and toxic materials was then concentratedto dryness in vacuo. This procedure was carried out at room temperatureand as quickly as possible to avoid hydrolysis of the active substanceby the acid conditions necessary for the water-insoluble solventtreatment.

For further purification, the aqueous concentrate was dried by heatingand the dried residue was dissolved in ml. of methanol. Undesiredmaterial was precipitated by pouring into two liters of acetone. Themethanol-acetone solution was ltered and in this way 61 gms. of solidscomprising inactive insoluble material was removed. The solutioncontaining the desired water-soluble oestrogenic substances was thenconcentrated to a small volume in vacuo and active principlesprecipitated by pouring into two liters of ether. The precipitate wasfiltered on and dried in vacuo. It weighed 29.9 gms. It containedwatersoluble oestrogenic activity equivalent to 20.5% sodium oestronesulphate measured by the modied Marrian-Kober test.

This represents 81% recovery of the oestrogenic potency of theconcentrate in a water-soluble form.

The resulting product is a pharmaceutically useful substance suitablefor clinical use on oral administration. It is more active whenadministered orally than free oestrone in alleviating the menopasalsyndrome in humans, and when administered to ovariectomized adult ratsin causing vaginal cornication. It is stable. It is substantiallyinsoluble in water-immiscible organic solvents, for example benzol andether. It is 10'0 cc. of the concentrate prepared as in Example 1 priorto the pH adjustment step to an acid Ipoint (p1-I 4) were taken todryness in vacuo and extracted with acetone. The acetone solution wasconcentrated to dryness in vacuo and the residue dissolved in Water andadjusted with 15% sulphuric acid to pH 4, and extracted with four equalvolumes of ethylene dichloride. The aqueous solution was adjsted to pI-I7, Washed with an equal volume of ethyl ether and the excess etherremoved in vacuo. The resulting aqueous concentrate contained over 90%of the original water-soluble oestrogenic activity f the startingconcentrate as measured by the modified Marrian-Kober test. It wasconcentrated in vacuo to dryness and yielded a bui colored amorphouspowder which was readily soluble in water and effectively free fromodoriferous substances. Emmple 3 To test for stability of theoestrogenic product obtained as described, 100 cc. of a 15% aqueousalcohol solution of the puried concentrate contained 600 Collip unitswere assayed orally on immature rats for its oestrogenic potency. Thesolution of the concentrate was stored in a stoppered 4 ounce glassbottle at 37 C. for a period of four months. During this period, thesolution was assayed repeatedly three times (end of rst month, secondmonth and fourth month). Within the error of the biological assay, therewas no evidence of deterioration.

It will be understood that the specific procedures and productsexemplified are susceptible to certain modication without departing fromthe spirit of the invention. The following remarks, although notexhaustive, will be of guidance to certain of the modications of whichthe subject matter is capable.

The products of this inventionare preferably prepared by rstconcentrating unhydrolyzed equine urine under conditions to preventhydrolysis so as to form a concentrate containing a water-solubleoestrogenic substance, extracting the concentrate with an organicWater-soluble solvent for the substance under conditions to preventhydrolysis so as to obtain an extract containing said substance, andconcentrating the extract under conditions to prevent hydrolysis.Preferably this process is eifected by absorbing an unhydrolyzed urinaryliquid with an adsorbent, preferably activated carbon, and maintaining amoderate temperature and a pH within a range from substantially neutralto mildly alkaline (preferably about '7 to about 8) and then elutingfrom the absorbent under similar conditions with an organicWater-soluble solvent for the substance.

AThe product resulting in a water-soluble substance insoluble in benzoland ether, and containing substantially all the water-solubleoestrogenic activity of the original urine and usually less than about2% of the original urinary solids. It contains no substantial quantityof free oestrogens. The product is stable. However, it still containsinactive and odoriferous substances and substances producing gastricdisturbances and nausea in some individuals.

In the washing or solvent extration step using a water-insolublesolvent, the pH range of from 6 2 to 6 is critical. Superior results areobtained? within the preferred range from 4 to 5. In order to hold anyhydrolysis to the absolute minimum it is desirable to operate rapidlyand at a low temperature, preferably within the range from 15 C. to 20C. and in any case not higher than 40 C. for any length of time. Thewashing o1' extraction step is usually repeated several, preferably 4 to6 times.

The extractants are substantially water-immiscible organic solvents forfats and oils, for phenols, for alkyl-substituted phenols, and forunconjugated sterols. 'I'he solvents having these specic characteristicsand effective for the purpose desired may be found among thehydrocarbons including the aliphatics such as hexane and higher, thearomatics such as benzene, toluene, etc.; the alicyclics such ascyclohexane; or the chlorinated hydrocarbons such as chloroform,ethylene dichloride, trichlorethylene, carbon tetrachloride orchlorobenzene. 1n summation, the essential characteristics required inthe solvent are that the Water-soluble, orally active oestrogenicconjugates be substantially insoluble in the solvent when operating inan acid environment; that the solvent be substantially water-insoluble;and that the solvent possesses substantial solvent action for fat, oilsand free phenolic compounds.

Of the solvents mentioned, chlorinated hydrocarbons are preferred,particularly ethylene dichloride, because the applicants have found thatsurprisingly little of the oestrogenic activity is extracted by thesesolvents. Moreover, these solvents are non-inflammable and thus betteradapted for large-scale processes.

The solution after extraction is neutralized to a pI- from substantiallyneutral to mildly alkaline. The applicants prefer to operate withintherange from 6 to 8.5 and more particularly from 7 to 7.5. Neutralizationmay be effected by*- the use of an alkali, preferably sodium hydroxide.Kor potassium hydroxide.

Washing the neutralized solution with an etheris an optional step, butthe applicants have found; it to be of considerable advantage in that itre-v moves any remaining water-immiscible solvent., Isopropyl or ethylether, or other solvent for the: water-immiscible solvent may beemployed.

The drying step is to remove 'water and to render the active materialsuitable for further treatment. One of the preferred methods of dryingis in vacuo at a temperature from about 40 C. to about 50 C.

The dried product is dissolved in a lower alcohol such as methyl alcoholand the undesired material is precipitated with an alcohol-soluble,ketonic solvent. As examples of ketonic solvents suitable for thepurpose may be mentioned loweralkyl ketones such as acetone, methylethyl ke-` tone, etc. The preferred ketone however is; acetone. toseparate out large amounts of ketone-insolubleV material Which theapplicants have found con--l tains little potency and to leaveketone-soluble material which the applicants have found con-4 tains thepotency. The step is carried out byadding an excess of the ketonicsolvent gradually, with stirring, until no further insoluble precipitateis being formed. Desirably, room temperature is maintained. Thesupernatant liquid y containing the oestrogenic activity is removed by'-ltration or centrifugation and is then concen-V` trated.

The resulting concentrate is dissolved in alco-.. hol and poured into anexcess of an ether, ethyl` Surprisingly, this step has been found,y

Lorisopropylietheribeing.preferred.l This forms-a= precipitatecontaining. the. oestrogenically active substance., theY ether-solubla4oils and-phenolic substances vremaining inthe supernatant liquid. Thepotent; precipitate. Vis separated and -preierably ,washedlwith ether,then dried.

Thproductoi the invention prepared as above has the, followingVcharacteristics:

Physical nature:

(a) VAs-a solid an amorphous powder (b)A As anaqueous solution.

The product contains aplurality of water-soluble oestrogenic substances,organic nitrogen com'- pounds, for example, indoxylsulphate and also a.substantial amount of- Valkaline-soluble fractions.

Stability:

Stable as a dry powder or in aqueous solution at room temperatureagainst auto-hydrolysis.

Aesthetic qualities:

Practically free from objectionable odour, and taste, and factors whichcause intestinal disturbancesV and nausea.

Pharmacological effect:

(a) More effective orally-than free -oestrone for alleviation rof themenopausal syndrome.

(b) More effective than oestrone sulphate in;

oral menopausal therapy.

(c) Free from toxicity at `therapeutic levels of dosage.

(d) Alleviates common autonom-ie and emotional symptoms accompanying themenopausal syndrome.

(e) Approximately two to two-andone-half times as active as theequivalent amount of free oestrone in producing vaginal corniiication`when administered orally to the adult ovariectomized rats.

Clinically, the product has a greater activitythan accounted for by theapparent sodium oes-v trone sulphate content as measured by the modifledMarrian-Kober test. (Colorirnetric estima tion) described in HTheEliminationof Estrin in Urine with the Photo-Electric Colorimeter by E.H. Venning et al. (J. B. C. 120, 225 (1937).

The hydrogen ion concentrations (pH) mentioned herein were measured onthe Beckman Laboratory model glass electrode pH meter .with nocorrection for metallic'ion concentration.

It will be understood that, without departing from the spirit of theinvention or the scope of the claims, various modications may be madeinthe specilc expedients described. The latter are illustrative and notoiered in a restricting sense, except as may be required by the state of'the prior art.

ici:

' lyzed equine urinary extract containing Watersolubleo'estrogenicactivity under substantially non-hydrolyzing conditions, acidifying saidconcentrate to within a pH range from about 2 to about 6, quicklyextracting the acidied aqueous material with a substantiallywater-immiscble solvent of the group consistngiof hydrocarbons andchlorinated hydrocarbons to cause separation of undesired material suchas oils-and phenols intothe solvent while leaving the desired,watersoluble oestrogenicallyl active principles in the aqueous phase,rapidly separating the aqueous phase'containing .the desired principlesin puriedand concentrated' form from the solvent fraction, and thenneutralizing the aqueous concenv:trate to a point where hydrolysis is ataminmum.

"solvent of the 4group consisting. of hydrocarbons andA chlorinatedhydrocarbons to cause separationV of undesired material such `as oilsHand phenols into the solventlwhile leaving thefdesired, water-solubleAoestrogenically active principles in theaqueousvphasarapidlyseparatingthe aqueous phase containing thedesired principles in purifiedand.V concentrated form from the. solvent fraction, and thenneutralizing the aqueous concentrate to. a pH of about 7 to about 8where hydrolysis is at a minimum.

3. The process of claiml; wherein the waterimrniscble solvent isethylene-dichloride.

4. The process of claim l; wherein the neutralizedV aqueous concentrateis extracted with diothyl ether.

5. A process for the preparation of a watersoluble oestrogenicallyactive product comprising, contacting substantially unhydrolyzedpregnant mares, urine with an adsorbent, eluating the desiredoestrogenic potency from the adsorbent, concentrating the eluate to forman aqueous concentrate having oestrogenic activity, said steps beingcarried out under conditions to avoid substantial hydrolysis, acidifyingsaid concentrate to within a pI-Irange of about 4 to about 6, quicklyextracting the acidied aqueous material with ethylene. dichloride inwhich undesired material such asoilsand phenols are soluble, rapidlyseparating the aqueousphase containing the desired principles from thesolvent fraction, neutralizing the purified aqueous concentrate to a pHin the neighborhood of about pH 7 to about 8, extracting theneutralizedproduct with ether and discarding thethe ether fraction,dissolving thev purified concentrate in a lower alcohol, adding analcohol-soluble, lower-alkyl ketone thus precipitating impurities fromthe alcoholic solution, and then concentrating the alcohol-ketonesolution to remove substantially all of the solvents leaving` aconcentrated oestrogenic fraction as the desired product.

A. ,STANLEY COOK. G. A. GRANT.

(References on following page) 9 10 REFERENCES CITED FOREGN PATENTSl'hef ftclnowin; rtelferences are of record in the. Number Country Datee 1S 'Pa en 459,536 Great Britain Jan. 11, 1937 5 Great Britain Jan. 15,OTHER REFERENCES Number Name Date 1,928,830 Dohrn Oct. 3, 1933 Katzmanin J. Biol. Chem. Nov. 1932, pages 2,190,248 Wollheim Feb. 13, 1940739-754, 2,196,295 Eberlein Apr. 9, 1940 10 New and Nonofcial Remedies,1945, page 440. 2,292,841 Necheles Aug. 11, 1942 2,363,549 RabnowitzNov. 28, 1944 2,429,398 Cook Oct. 21, 1947

1. A PROCESS FOR THE PREPARATION OF A WATERSOLUBLE OESTROGENICALLYACTIVE COMPOSITION COMPRISING CONCENTRATING A SUBSTANTIALLY UNHYDROLYZEDEQUINE URINARY EXTRACT CONTAINING WATERSOLUBLE OESTROGENIC ACTIVITYUNDER SUBSTANTIALLY NON-HYDROLYZING CONDITIONS, ACIDIFYING SAIDCONCENTRATE TO WITHIN A PH RANGE FROM ABOUT 2 TO ABOUT 6, QUICKLYEXTRACTING THE ACIDIFIED AQUEOUS MATERIAL WITH A SUBSTANTIALLYWATER-IMMISCIBLE SOLVENT OF THE GROUP CONSISTING OF HYDROCARBONS ANDCHLORINATED HYDROCARBONS TO CAUSE SEPARATION OF UNDESIRED MATERIAL SUCHAS OILS AND PHENOLS INTO THE SOLVENT WHILE LEAVING THE DESIRED,WATERSOLUBLE OESTROGENICALLY ACTIVE PRINCIPLES IN THE AQUEOUS PHASE,RAPIDLY SEPARATING THE AQUEOUS PHASE CONTAINING THE DESIRED PRINCIPLESIN PURIFIED AND CONCENTRATED FORM FROM THE SOLVENT FRACTION, AND THENNEUTRALIZING THE AQUEOUS CONCENTRATE TO A POINT WHERE HYDROLYSIS IS AT AMINIMUM.